In high resolution scanning systems, focusing is a major rate-limiting step in that it is not unusual to find systems which spend approximately half of the imaging time on focusing. Thus, optimizing the focusing process is important for providing system throughput. For fluorescent imaging in particular, the situation is more extreme, as will be explained below.
Conventional microscopy focus algorithms rely on the images themselves. A series of images at different focal planes is examined and the image with the largest amount of detail, or the greatest amount of information, is selected as being correctly focused. However, current systems are unable to distinguish between genuine details and noise, and thus images with high signal to noise ratio (SNR) are required for effective focusing.
Considering conventional focusing in greater detail, conventional focusing relies on the quality of the images. In order to perform the focus sequence, the system takes a series of images around an estimated focus position. For each image, the system records its position, and computes a focus score which characterizes the sharpness of the respective image. In a final step, the system computes a position for which the focus score is maximal, and the computed position is taken as the focus position.
The sharpness function is one of many functions that evaluate the amount of fine detail in an image. A good sharpness function is sensitive (a small change in the amount of detail produces a large change in the focus score), and well-behaved over a large range. That is to say, as the distance from the focus position increases, the focus score decreases, even for large distances from the focus position. Considering by way of example the following sequence:                1. take an image,        2. convolve the image with a fine detail filter (e.g. Sobel filter),        3. sum the absolute value of the intensity of pixels in the convolved image, and        4. set the focus score (FS) to the above sum.        
The above sequence is repeated typically five to ten times for each one of five to ten different images and a focus position is computed, preferably by interpolation between the images to find a maximal sum position.
The main drawback to the above-described sequence, and a drawback which applies to a complete family of functions, is that they are sensitive to noise. Thus, unless image quality is good, the largest contribution for the focus score comes from the noise, and both the requirements for sensitivity and for a large range are lost
Fluorescent imaging is characterized as being a low light application, meaning that only low levels of light are emitted from the sample. In addition, high resolution is needed in order to distinguish details of the sample. Thus a fluorescent imaging system is required to provide large magnification of the sample, to have a high capacity for collecting light and requires relatively long exposure times in order to obtain a reasonable image. In order to provide the high magnification necessary, high magnification objective lenses are used, typically of ×40 and above. In order to achieve the high resolution and to collect as much light as possible, objective lenses with high numerical apertures (NA), typically 0.75 and above, are generally used. Typical exposure times of the order of a second and above are needed in order to obtain high quality, low noise images.
It will be appreciated that a high NA causes a low depth of field. Thus even slight deviations in the distance between the objective and the sample can lead to severe misfocusing of the image. Conventional focusing of the kind described above typically requires five-ten images. The requirement of low noise images means that the exposure time used for focus need to be similar to that used for the actual imaging. In the case of fluorescent imaging, a focusing time of around five-ten seconds is therefore implied. Thus in fluorescent imaging the system spends the vast majority of its time focusing, and the focusing problem is an obstacle to providing a high throughput fluorescent imaging system.